The phylogeny of 116 species and varieties of Pythium was studied using parsimony and phenetic analysis of the ITS region of the nuclear ribosomal DNA. The D1, D2 and D3 regions of the adjacent large subunit nuclear ribosomal DNA of half the Pythium strains were also sequenced and gave a phylogeny congruent with the ITS data. All the 40 presently available ex-type strains were included in this study, as well as 20 sequences of recently described species from GenBank. Species for which no ex-type strains were available were represented by either authentic strains (6), strains used in the 1981 monograph of the genus by van der Plaats-Niterink (33), or strains selected on morphological criteria (17). Parsimony analysis generated two major clades representing the Pythium species with filamentous or globose sporangia. A small clade of species with contiguous sporangia was found in between the two main clades. A total number of 11 smaller clades was recognized, which often correlated with host-type or substrate and in several cases with a subset of morphological characters. Many characters used in species descriptions, such as antheridium position, did not correlate with phylogeny. A comparison of the ex-type and representative strains with all ITS sequences of Pythium in GenBank revealed limited infraspecific variation with the exception of P. rostratum, P. irregulare, P. heterothallicum, and P. ultimum. The total number of species examined was 116 (including 60 ex-type strains). Twenty-six species had ITS sequences identical or nearly identical to formerly described species, suggesting possible conspecificity. The importance of comparing ITS sequences of putative new species to the now available ITS database in order to avoid unwarranted new species names being introduced.